Whole Genome Amplification in Preimplantation Genetic Testing in the Era of Massively Parallel Sequencing

Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Successful whole genome amplification (WGA) is a cornerstone of contemporary preimplantation genetic testing (PGT). Choosing the most suitable WGA technique for PGT can be particularly challenging because each WGA technique performs differently in combination with different downstream processing and detection methods. The aim of this review is to provide insight into the performance and drawbacks of DOP-PCR, MDA and MALBAC, as well as the hybrid WGA techniques most widely used in PGT. As the field of PGT is moving towards a wide adaptation of comprehensive massively parallel sequencing (MPS)-based approaches, we especially focus our review on MPS parameters and detection opportunities of WGA-amplified material, i.e., mappability of reads, uniformity of coverage and its influence on copy number variation analysis, and genomic coverage and its influence on single nucleotide variation calling. The ability of MDA-based WGA solutions to better cover the targeted genome and the ability of PCR-based solutions to provide better uniformity of coverage are highlighted. While numerous comprehensive PGT solutions exploiting different WGA types and adjusted bioinformatic pipelines to detect copy number and single nucleotide changes are available, the ones exploiting MDA appear more advantageous. The opportunity to fully analyse the targeted genome is influenced by the MPS parameters themselves rather than the solely chosen WGA.publishersversionPeer reviewe

The association of FMR1 gene (CGG)n variation with idiopathic female infertility

Introduction: The FMR1 gene plays an important role in brain development and in the regulation of ovarian function. The FMR1 gene contains CGG repeat variation and the expansion of the repeats is associated with various phenotypes e.g. fragile X syndrome, premature ovarian failure, etc. Repeats ranging 0.05). Conclusions: In our study, the FMR1 gene high-normal alleles were associated with secondary infertility. However, to address the controversies related to the role of FMR1 genes in the development of diminished ovarian reserve, further studies on the subject are required.publishersversionPeer reviewe

Phenotypic variability and diagnostic characteristics in inherited peripheral neuropathy in Latvia

Publisher Copyright: © Latvian Academy of Sciences.Inherited peripheral neuropathies (IPN) are a clinically and genetically heterogeneous group of disorders. The most common IPN is Charcot-Marie-Tooth (CMT) disease. Here we describe IPN clinical variability and diagnostic characteristics in the Latvian population. A total of 101 patients were enrolled in the study. Genetic testing consisted of PMP22 copy number analysis and whole-exome sequencing (WES). Clinical assessment comprised CMT Neuropathy Score version 2 (CMTNSv2), CMT Examination Score, pain, anxiety and memory/cognitive ability testing. The diagnostic yields for PMP22 copy number detection and WES were 45.8% and 77.8%, respectively. Disease severity assessment indicated high clinical heterogeneity, with CMTNSv2 scores ranging between 0 and 33. More than one-third of patients reported pain, and it was found to be significantly more common in patients with at least a mild anxiety level. From the initial development of symptoms, on average, it took more than 13 years for a diagnosis of IPN to be confirmed. This study updates the IPN genetic and clinical profile of the Latvian population and demonstrates the presence of a high level of heterogeneity. The time to diagnosis for IPN patients needs to be improved by employing multiplex ligation-dependent probe amplification initially followed by WES.publishersversionPeer reviewe

Cytokine Response Following SARS-CoV-2 Antigen Stimulation in Patients with Predominantly Antibody Deficiencies

Funding Information: This research was funded by the Latvian Council of Science project Nr.lzp-2020/1-0269. Publisher Copyright: © 2023 by the authors.Predominantly antibody deficiencies (PADs) are inborn disorders characterized by immune dysregulation and increased susceptibility to infections. Response to vaccination, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), may be impaired in these patients, and studies on responsiveness correlates, including cytokine signatures to antigen stimulation, are sparse. In this study, we aimed to describe the spike-specific cytokine response following whole-blood stimulation with SARS-CoV-2 spike peptides in patients with PAD (n = 16 with common variable immunodeficiency and n = 15 with selective IgA deficiency) and its relationship with the occurrence of coronavirus disease 2019 (COVID-19) during up to 10-month follow-up period. Spike-induced antibody and cytokine production was measured using ELISA (anti-spike IgG, IFN-γ) and xMAP technology (interleukin-1β (IL-1β), IL-4, IL-6, IL-10, IL-15, IL-17A, IL-21, TNF-α, TGF-β1). No difference was found in the production of cytokines between patients with PAD and controls. Anti-spike IgG and cytokine levels did not predict contraction of COVID-19. The only cytokine that distinguished between vaccinated and naturally infected unvaccinated PAD patients was IFN-γ (median 0.64 (IQR = 1.08) in vaccinated vs. 0.10 (IQR = 0.28) in unvaccinated). This study describes the spike-specific cytokine response to SARS-CoV-2 antigens, which is not predictive of contracting COVID-19 during the follow-up.publishersversionPeer reviewe

Management of Pregnancy with Cervical Shortening: Real-Life Clinical Challenges

Funding Information: The study was funded by the Fundamental and Applied Research Projects grant of The Latvian Council of Science (Project No. 2020/1-0042 to DR). The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: © 2023 by the authors.Background and Objectives: Preterm birth is the leading cause of neonatal mortality worldwide and may be responsible for lifelong morbidities in the survivors. Cervical shortening is one of the common pathways to preterm birth associated with its own diagnostic and management challenges. The preventive modalities that have been tested include progesterone supplementation and cervical cerclage and pessaries. The study aimed to assess the management strategies and outcomes in a group of patients with a short cervix during pregnancy or cervical insufficiency. Materials and Methods: Seventy patients from the Riga Maternity Hospital in Riga, Latvia, were included in the prospective longitudinal cohort study between 2017 and 2021. Patients were treated with progesterone, cerclage, and/or pessaries. The signs of intra-amniotic infection/inflammation were assessed, and antibacterial therapy was given when the signs were positive. Results: The rates of PTB were 43.6% (n = 17), 45.5% (n = 5), 61.1% (n = 11), and 50.0% (n = 1) in progesterone only, cerclage, pessary, and cerclage plus pesssary groups, respectively. The progesterone therapy was associated with a reduced preterm birth risk (x2(1) = 6.937, p = 0.008)), whereas positive signs of intra-amniotic infection/inflammation significantly predicted the risk of preterm birth (p = 0.005, OR = 3.82, 95% [CI 1.31–11.11]). Conclusions: A short cervix and bulging membranes, both indicators of intra-amniotic infection/inflammation, are the key risk factors in preterm birth risk predictions. Progesterone supplementation should remain at the forefront of preterm birth prevention. Among patients with a short cervix and especially complex anamnesis, the preterm rates remain high. The successful management of patients with cervical shortening lies between the consensus-based approach for screening, follow-up, and treatment on the one side and personalising medical therapy on the other.publishersversionPeer reviewe

Allelic variants of breast cancer susceptibility genes PALB2 and RECQL in the Latvian population

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Performance comparison of two whole genome amplification techniques in frame of multifactor preimplantation genetic testing

Publisher Copyright: © 2018, The Author(s).Purpose: To compare multiple displacement amplification and OmniPlex whole genome amplification technique performance during array comparative genome hybridization (aCGH), Sanger sequencing, SNaPshot and fragment size analysis downstream applications in frame of multifactor embryo preimplantation genetic testing. Methods: Preclinical workup included linked short tandem repeat (STR) marker selection and primer design for loci of interest. It was followed by a family haplotyping, after which an in vitro fertilization preimplantation genetic testing (IVF-PGT) cycle was carried out. A total of 62 embryos were retrieved from nine couples with a confirmed single gene disorder being transmitted in their family with various inheritance traits—autosomal dominant (genes—ACTA2, HTT, KRT14), autosomal recessive (genes—ALOX12B, TPP1, GLB1) and X-linked (genes—MTM1, DMD). Whole genome amplification (WGA) for the day 5 embryo trophectoderm single biopsies was carried out by multiple displacement amplification (MDA) or polymerase chain reaction (PCR)-based technology OmniPlex and was used for direct (Sanger sequencing, fragment size analysis, SNaPshot) and indirect mutation assessment (STR marker haplotyping), and embryo aneuploidy testing by array comparative genome hybridization (aCGH). Results: Family haplotyping revealed informative/semi-informative microsatellite markers for all clinical cases for all types of inheritance. Indirect testing gave a persuasive conclusion for all embryos assessed, which was confirmed through direct testing. The overall allele dropout (ADO) rate was higher for PCR-based WGA, and MDA shows a better genomic recovery scale. Five euploid embryos were subjected to elective single embryo transfer (eSET), which resulted in four clinical pregnancies and birth of two healthy children, which proved free of disease causative variants running in the family postnataly. Conclusions: A developed multifactor PGT protocol can be adapted and applied to virtually any genetic condition and is capable of improving single gene disorder preimplantation genetic testing in a patient-tailored manner thus increasing pregnancy rates, saving costs and increasing patient reliability.publishersversionPeer reviewe

Clinical Phenotyping and Biomarkers in Spinal and Bulbar Muscular Atrophy

Funding Information: HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018-02532), the European Research Council (#681712), Swedish State Support for Clinical Research (#ALFGBG-720931), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809-2016862), the UK Dementia Research Institute at UCL, and Riga Stradins University Internal grant No. 131005. Publisher Copyright: © Copyright © 2021 Millere, Rots, Glazere, Taurina, Kurjane, Priedite, Gailite, Blennow, Zetterberg and Kenina.Background: Spinal and bulbar muscular atrophy (SBMA) or Kennedy disease [OMIM: 313200] is a rare X-linked neuromuscular disease. Patients commonly present with muscle cramps, tremors, leg weakness, dysarthria and dysphagia. Methods: We deeply phenotyped and evaluated the possible extent of affected systems in all patients with SBMA in Latvia (n = 5). In addition, neurophysiological studies and blood analyses were used to perform a molecular diagnosis and evaluate biochemical values. We analyzed neurofilament light (NfL) as a possible biomarker. Results: Neurological examination revealed typical SBMA clinical manifestations; all patients had small or large nerve fiber neuropathy. Three of five patients had increased neurofilament light levels. Conclusion: The study confirms the systemic involvement in patients suffering from SBMA. Increased NfL concentration was associated with either peripheral neuropathy or decreased body mass index. The complex phenotype of the disease should be kept in mind, as it could help to diagnose patients with SBMA.publishersversionPeer reviewe

Role of Single Nucleotide Variants in FSHR, GNRHR, ESR2 and LHCGR Genes in Adolescents with Polycystic Ovary Syndrome

Funding Information: Funding: The study was financially supported by a Riga Stradins University internal research grant. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Background: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women, affecting up to 16.6% of reproductive-age women. PCOS symptoms in adolescents comprise oligomenorrhoea/amenorrhoea and biochemical and/or clinical hyperandrogenism. Long-term health risks of PCOS patients include infertility, metabolic syndrome, type 2 diabetes and cardiovascular disease. Genetic factors have been proven to play a role in development of the syndrome and its symptoms. Objective: To investigate single nucleotide variants (SNVs) in the GNRHR, ESR2, LHCGR and FSHR genes in adolescent patients with PCOS and their association with PCOS symptoms. Methods: We conducted a cross-sectional study comprising of 152 adolescents: 63 patients with PCOS, 22 patients at risk of developing PCOS and 67 healthy controls. Participants were recruited from out-patients attending a gynaecologist at the Children’s Clinical University Hospital, Riga, Latvia, between January 2017 and December 2020. Genomic DNA was extracted from whole blood, and SNVs in the GNRHR, ESR2, LHCGR and FSHR genes were genotyped. The distributions of SNV genotypes were compared among the three groups and genotype-phenotype associations within the PCOS group were evaluated. Results: No statistically significant differences were found in the distributions of genotypes for GNRHR (rs104893837), ESR2 (rs4986938), LHCGR (rs2293275) and FSHR (rs6166, rs6165, rs2349415) among PCOS patients, risk patients and healthy controls. Within the PCOS group, ESR2 rs4986938 minor allele homozygous patients had a significantly higher level of total testosterone than major allele homozygous patients and heterozygous patients. A significantly higher total testosterone level was also observed in PCOS patients carrying the LHCGR rs2293275 minor allele compared with major allele homozygous patients. Conclusions: The SNVs ESR2 rs4986938 and LHCGR rs2293275 play a role in the phenotypic characteristics of PCOS. To fully uncover their influence on the development of PCOS and its symptoms, further studies of larger cohorts and a follow up of this study sample through to adulthood are required. Furthermore, studies of adolescent PCOS patients conducted prior to the latest European Society of Human Reproduction and Embryology (ESHRE) criteria (2018) should be re-evaluated as the study groups might include risk patients according to these updated criteria, thereby potentially significantly impacting the published results.publishersversionPeer reviewe

Genetic distinctiveness of rye in situ accessions from Portugal unveils a new hotspot of unexplored genetic resources

Rye(SecalecerealeL.)is a cereal crop of major importance in many parts of Europe and rye breeders are presently very concerned with th e restrict pool of rye genetic resources available.Such narrowing of rye genetic diversity results from the presence of“Petkus ”pool in most modern rye varieties as well as “Petkus”דCarsten” heterotic pool in hybrid rye breeding programs.Previous studies on rye’s genetic diversity revealed more over a common genetic background on landraces(exsitu) and cultivars,regardless of breeding level or geographical origin.Thus evaluation of in situ populations is of utmost importance to unveil “onfarm” diversity,which is largely undervalued.Here,we perform the first comprehensive assess ment of rye’s genetic diversity and population structuring using cultivars,exsitu landraces along a comprehensive sampling of insitu accessions from Portugal,through amolecular-directed analysis using SSRs markers.Rye genetic diversity and population structure analysis does not present any geographical trend but disclosed marked differences between genetic backgrounds of in situ accessions and those of cultivars/exsitu collections.Such genetic distinctiveness of insitu accessions highlights their unexplored potential as new genetic resources,which can be used to boost rye breeding strategies and the production of new varieties.Overall,our study successfully demonstrates the high prospective impact of comparing genetic diversity and structure of cultivars,exsitu,and in situ samples in ascertaining the status of plant genetic resources (PGR)info:eu-repo/semantics/publishedVersio